Meningitis vaccine

ABSTRACT

A high molecular weight capsular polysaccharide from H. influenza type b is an effective immunogenic agent against infection caused by the H. influenza type b bacterium.

RELATED APPLICATION

The present application is a continuation-in-part of co-pendingapplication Ser. No. 002,818 filed on Jan. 12, 1979.

BACKGROUND OF THE INVENTION

Hemophilus influenza type b is the most important cause of bacterialmeningitis in children between the ages of 3 months and 4 years. Theeffect of bacterial meningitis in infants and young children can besevere and may include pyarthrosis, epiglottitis, maxillary sinusitis,ethmoid sinusitis, pneumonia and otitis media. Pyarthrosis in infants isdifficult to diagnose and can lead to irreversible damage in theadvanced lesion stage; epiglottitis can occur as a result of bacteremiaand a tracheotomy is usually mandatory because of the high probabilityof sudden death as a result of respiratory or cardiac arrest; maxillarysinusitis may remain in a chronic condition for months if untreatedwhile ethmoid sinusitis is very acute leading to periorbital cellulitisin one or both eyes. Pneumonia and otitis media can occur also as aconsequence of nasopharyngeal involvement. The possibility of asynergistic effect of bacteria plus virus may exist and untreated casesmay terminate fatally. Despite the effectiveness of certainantimicrobial therapy, no decline in mortality has been observed duringthe last decade to type b-induced meningitis while recovery from thedisease has imposed a high percentage of neurological impairment.Clinically, there appears to be an increase in the number of infantsunder 2-3 months of age who are susceptible as well as in olderchildren; increased adult susceptibility also may be a problem.

OBJECTS OF THE INVENTION

It is, accordingly, an object of the present invention to provide avaccine which is effective against H. influenza type b. Another objectis to provide an antigenic and immunogenic high molecular weightcapsular polysaccharide from H. influenza type b. A further object is toprovide compositions for administering the antigenic and immunogenichigh molecular weight capsular polysaccharide as a vaccine. Stillanother object is to provide a method for preparing the antigenic andimmongenic high molecular weight capsular polysaccharide. These andother objects of the present invention will be apparent from thefollowing description.

SUMMARY OF THE INVENTION

A high molecular weight capsular polysaccharide from H. influenza type bis an effective immunogenic agent against infection caused by the H.influenza bacterium. Substantially all of the polysaccharide has amolecular weight of at least about 5,000,000 daltons and about half ofthe polysaccharide has a molecular weight of about 10,000,000 daltons.

DETAILED DESCRIPTION

The present invention is directed to a high molecular weigh capsularpolysaccharide prepared from an encapsulated strain of H. influenza typeb. Substantially all of this capsular polysaccharide is composed of highmolecular weight polysaccharide. Substantially all of the polysaccharidehas a molecular weight of at least about 5,000,000 daltons and abouthalf of the polysaccharide has a molecular weight of at least about10,000,000 daltons. The polysaccharide of the present invention consistsof about equal parts of ribose, ribitol and phosphate, and has thegeneral formula

    [Ribose-Ribitol-PO.sub.4 ].sub.n

The high molecular weight capsular polysaccharide of the presentinvention is obtained from an encapsulated strain of H. influenza typeb. A culture of the organism is grown under suitable conditions in anutrient medium. After completion of the fermentation the cell paste isremoved, e.g., by centrifugation and the remaining fluid is treated toprecipitate unwanted proteins, nucleic acids and lipopolysaccharides. Alow concentration, i.e., up to about 0.1% of cetrimonium bromide issuitable for this precipitation. The precipitates are removed, e.g., bycentrifugation, and the supernatent fluid is concentrated to aboutone-tenth or less of the original volume. Preferably, the concentrationis effected by ultrafiltration whereby low molecular weightcontaminants, i.e., below about 50,000 molecular weight are alsoremoved.

A water-miscible alcohol is added to the concentrated liquid toprecipitate nucleic acids and other alcohol insoluble contaminants. Thealcohol is preferably ethanol which is added until the ethanolconcentration is from about 9.5% to about 10.5% (vol/vol), therebyprecipitating nucleic acids and other alcohol insoluble contaminants.The precipitates are removed and discarded. An additional quantity of awater miscible alcohol is added to the concentrated liquid toprecipitate the high molecular weight capsular polysaccharide of thepresent invention. The alcohol is preferably ethanol which is addeduntil the ethanol concentration to from about 19% to about 21% (vol/vol)thereby precipitating the high molecular weight capsular polysaccharideof the present invention.

The alcohol-containing liquid is separated from the precipitatedpolysaccharide, e.g., by centrifugation, filtration, settling,aspiration or other suitable means, and the latter is extracted with anaqueous solution of CaCl₂, typically in a concentration of from about0.1 M to about 2.0 M preferably about 1.0 M to enhance separation of thecapsular polysaccharide from impurities. A water-miscible alcohol,preferably ethanol in concentration from about 9.5% to about 10.5%(vol/vol) is added to the CaCl₂ extract thereby precipitating anyremaining nucleic acids and other alcohol insoluble contaminants. Thealcohol-containing liquid is separated from the precipitates, e.g., bycentrifugation, filtration, settling, aspiration or other suitablemeans. The precipitates are discarded. Any lipid-like floating materialin the liquid is removed by any suitable means, e.g., by filtering theliquid or by decanting it through cheese cloth. An additional quantityof a water-miscible alcohol, is added, preferably ethanol to aconcentration above about 50% (vol/vol), most preferably to from about70 to about 80% (vol/vol) thereby precipitating the high molecularweight capsular polysaccharide of the present invention.

The precipitate is separated from the alcohol-containing liquid, e.g. byfiltration or centrifugation, washed and dried. The dried product isresuspended in sodium acetate solution, pH 6.5-7.4, and extracted withphenol to remove any residual protein contaminants. The aqueous phase istreated, e.g. by dialysis or dilution, to reduce the phenolconcentration to below about 1%. To the treated aqueous phase, CaCl₂ isadded to a concentration of from about 0.01 M to about 0.1 M, preferablyabout 0.05 M, followed by slow addition of a water-miscible alcohol,preferably ethanol to a final concentration of above 50% (vol/vol), mostpreferably to from about 70 to about 80% (vol/vol) to precipitate thecapsular polysaccharide. The resulting precipitate is collected, washedand dried.

The dried material is resuspended in a dilute aqueous solution of CaCl₂,typically from about 0.01 M to about 0.1 M, preferably about 0.05 M, andcentrifuged (at about 100,000×G) until over half of the protein andendotoxin contaminants are contained in a glassine pellet. The pellet isdiscarded and a water-miscible alcohol is added slowly to thesupernatant liquid. Preferably the alcohol is ethanol added in quantityeffective to raise the ethanol concentration to above 50% (vol/vol),preferably to from about 70 to about 80% (vol/vol). The resultingprecipitate is collected, e.g., by centrifugation, filtration ordecanting, and washed and dried.

The dried material is resuspended in a dilute aqueous solution of CaCl₂,typically from about 0.01 M to about 0.1 M, preferably about 0.05 M, anda water-miscible alcohol is added slowly to the solution. Preferably thealcohol is ethanol which is added in quantity effective to raise thefinal concentration of ethanol to from about 9 to about 11% (vol/vol).The mixture is clarified by centrifugation at about 100,000×G and thepellet is discarded. Additional water-miscible alcohol is added,preferably ethanol, in quantity effective to bring the ethanolconcentration above 50% (vol/vol), preferably to from about 70 to about80% (vol/vol). After complete precipitation, the polysaccharideprecipitate is collected by centrifugation, and washed and dried.

Pharmaceutical compositions for administering the antigenic andimmunogenic high molecular weight polysaccharide as a meningitis vaccinecan be prepared by dissolving the purified polysaccharide in aphysiologically acceptable medium such as isotonic saline or PBS(phosphate buffered saline) solution which may contain an appropriateamount of a preservative such as thimerosal or phenol according tomethods known in the art.

The vaccine so prepared can be used in single dosage or multiple formsto afford sufficient protection against infection caused by the H.influenza bacterium in humans, especially in children. The single dosagelevel is from 10 μg to 100 μg, preferably 25-50 μg for adults; 10-25 μgfor children.

Following two such injections given at two to eight week intervals,preferably four weeks, protective immunity can be sustained for up totwo years.

The following examples illustrate the present invention without,however, limiting the same thereto. All temperatures are expressed indegrees Celsius.

EXAMPLE 1

A culture of a pathogenic isolate of H. influenza type b, e.g., Rossstrain, is grown in a medium in which yeast extract dialysate is used inplace of whole yeast extract. After the fermentation is completed, thecell paste is removed by Sharples centrifugation and to the supernatentfluid (180 liters) there is added cetrimonium bromide to a concentrationof 0.1 weight %. The resulting precipitate is removed by a secondSharples centrifugation at 45,000 rpm (60,000×G) at 20° and discarded.The supernatant fluid is collected and concentrated at 4° C. on anAmicon DC-30 unit with XM-50 (50,000 Daltons cut-off) hollow fibercartridges (4.5 m² membrane area; 2.0 lmp air flow at 20 psi;concentration=10 liters/hour). After 24 hours the original 180 liters ofsupernatant fluid are reduced in volume to 10.0 liters of retentate. Thefiltrate is discarded.

To the XM-50 retentate (10.0 liters), 1.11 liters of 100% ethanol isadded dropwise with stirring at 4° to a final concentration of 10%ethanol by volume. The mixture is allowed to stir 2-3 additional hoursand to stand 12-18 hours at 4° to insure complete precipitation. Thesupernatant fluid is collected by aspiration and finally bycentrifugation in the Beckman J-21B at 11,000×G (8,000 rpm in the JA-10rotor) for 30 minutes at 4°. The insoluble pellet is discarded. To the10% ethanol soluble fraction (11.0 liters) 1.375 liters of 100% ethanolis added dropwise with stirring to a final concentration of 20% byvolume. The mixture is allowed to stir 2-3 additional hours and to stand12-18 hours at 4° to insure complete precipitation.

The resulting 10% ethanol soluble, 20% ethanol insoluble precipitate iscollected by centrifugation in the Beckman J-21B at 11,000×G (8,000 rpmin the JA-10 rotor) for 30 minutes at 4°. The 20% ethanol supernatantfluid is discarded and 30.0 g of the resultant viscous precipitate(Yield, 45.0 g), is mixed with 170.0 ml cold glass-distilled H₂ O and200.0 ml of cold 2 M CaCl₂.2H₂ O is added. The mixture (finalconcentration=1.0 M CaCl₂) is extracted in an ice:water bath at settingNo. 2 in the Omnimixer for 30 minutes. Finally, an additional 100.0 mlof 1 M CaCl₂ is added and extraction continued for 15 minutes longer.The CaCl₂ extract (500 ml) is brought to 10% ethanol by adding 55.5 mlof 100% ethanol dropwise with stirring at 4°. After additional stirringfor 2-3 hours, the mixture is allowed to stand 12-18 hours at 4° toinsure complete precipitation. The mixture is centrifuged in the BeckmanJ-21B at 11,000×G (8,000 rpm in the JA-10 rotor) for 30 minutes at 4°.The supernatant fluid is decanted through cheese cloth to removelipid-like floating material. The insoluble pellet is discarded and the10% ethanol soluble supernatant fluid (500 ml) is brought to 75% ethanolby adding 1,300 ml of 100% ethanol dropwise with stirring over a 2-3hour period. The mixture is then allowed to stand 12-18 hours at 4° toinsure complete precipitation.

The resulting insoluble precipitate is recovered on a Buchner funnelwith a medium glass fritted disc (pore size 10-15 microns), washed threetimes with 100% ethanol (250 ml per wash) and three times with acetone(250 ml per wash). All washes are discarded. The insoluble product isdried in vacuo over anhydrous CaCl₂ at 4°. The yield is 10.0 grams.

Four (4.0) grams of the dried insoluble product are resuspended in 400ml (10 mg/ml) of 0.488 M sodium acetate pH 6.9 with the aid of a Douncehomogenizer. The sodium acetate solution is immediately extracted threetimes with 200 ml each of a fresh aqueous phenol solution made asfollows: 180 ml of 0.488 M sodium acetate pH 6.9 is added to a one poundbottle of crystalline phenol and mixed until a complete solution iseffected. Each phenol extract is centrifuged for 30 minutes at 11,000×Gat 4° in the Beckman J-21B to break the emulsion. The aqueous phases arepooled and extracted two (2) additional times with phenol solution in asimilar manner. The phenol phases are discarded and the pooled aqueousphases (500 ml) are dialyzed at 4° for 22 hours with three 14.0 literchanges of glass distilled H₂ O. The final dialysis ratio is 1:22,000volumes. No trace of phenol odor is present in the final dialysate.

To the dialysate (500 ml) there is added 12.5 ml of 2.0 M CaCl₂ to afinal concentration of 0.05 M CaCl₂. The solution is then brought to 75%ethanol with dropwise addition over 2-3 hours of 1,500 ml of 100%ethanol to the rapidly stirring solution. After standing 12-18 hours at4° the resultant precipitate is collected by centrifugation in theBeckman J-21B at 27,000×G (15,000 rpm in the JA-20 rotor) at 4° for 30minutes, washed two times with 100% ethanol and two times with acetone,and dried in vacuo over anhydrous CaCl₂ at 4°. The yield is 1.0 gram.

The dried material (1.0 g) is resuspended at 5 mg/ml in 200 ml of 0.05 MCaCl₂ and centrifuged at 100,000×G (40,000 rpm in the 50.2 Ti rotor) for90 minutes at 4° in the Beckman L2-75 centrifuge. The clear supernatantfluid (195 ml) is decanted off the small glassine pellet and brought to75% ehtanol with dropwise additiona of three volumes of 100% ethanol(585 ml ethanol) over a period of 2-3 hours, to the rapidly stirringsolution. After standing 12-18 hours at 4° the precipitate is collectedby centrifugation (27,000×G) washed two times with 100% ethanol, twotimes with acetone and dried in vacuo over anhydrous CaCl₂ at 4°. Theyield is 802.0 mg. The pellet is discarded.

The dried material (785 mg) is resuspended in 180 ml of 0.05 M CaCl₂(4.4 mg/ml and brought to 10% ethanol with dropwise addition of 20.0 ml100% ethanol. The mixture is clarified immediately by centrifugation at100,000×G (40,000 rpm in the 50.2 Ti rotor) for 90 minutes at 4°. Thepellet is discarded and the clear supernatant fluid (194 ml) is broughtto 75% ethanol by adding 505 ml of 100% ethanol. After stirring 2-3hours the mixture is left at 4° for 12-18 hours to insure completeprecipitation.

The polysaccharide precipitate is collected by centrifugation and washedtwo times with 100% ethanol and two times with acetone. After drying invacuo over anhydrous CaCl₂, the yield is 724.63 mg of a polysaccharidehaving the following characterization:

    ______________________________________                                        Ribose     =      28.%        (Orcinol)                                       Nucleic acid                                                                             =      0.8%        (Optical Density)                               Protein    =      1.0%        (Lowry)                                         Kav        =      0.01%       (52%), 0.19 (48%)                                                             (Sepharose 4B)                                  TGA        =      12.2%                                                       Limulus (0.0005 γ/ml) = negative                                        Rabbit pyrogen (0.25 γ/kg) = negative                                   Immunodiffusion: No Con A reactivity (at 1.0 mg/ml)                           Reaction of identity with polyribophosphate                                    vs. anti-H. influenza type b (at 4.0 μg/ml)                               Back-extrapolated theoretical yield = 2887.25                                 mg/200 liter fermenter, or 14.43 mg/liter of culture                          medium.                                                                       ______________________________________                                    

EXAMPLE 2

The purified polysaccharide is dissolved in PBS (phosphate bufferedsaline) at a concentration of 20 μg/ml and stored with a sufficientamount of phenol (preservative) at -20° C. until use. Children andinfants are immunized by an intramuscular injection of 0.5 ml containing10 μg of the purified polysaccharide. A booster injection of 0.5 ml alsocontaining 10 μg of the purified polysaccharide is given intramuscularlyfour (4) weeks after the primary immunization.

EXAMPLE 3

The purified polysaccharide is dissolved in PBS (phosphate bufferedsaline) at a concentration of 100 μg/ml. The mixture is then sterilefiltered and stored at -20° C. until use. Adults, especially females ofchild-bearing age (in order to protect offspring from neonatalmeningitis caused by H. Influenza type b) are immunized by anintramuscular injection of 0.5 ml containing 50 μg of the purifiedpolysaccharide.

What is claimed is:
 1. An antigenic and immunogenic capsularpolysaccharide from H. influenza type b consisting of about equal partsof ribose, ribitol and phosphate, substantially all of thepolysaccharide having a molecular weight of at least about 5,000,000,and about half of the polysaccharide having a molecular weight of atleast about 10,000,000.